The Hsp90 binding protein FKBP52, through interactions with the steroid hormone receptors, plays important physiological and potentially pathological roles in mammals. FKBP52 specifically regulates the androgen, progesterone and glucocorticoid receptors. Thus, FKBP52 represents an attractive therapeutic target for the treatment of hormone-dependent cancers such as prostate cancer. We have made progress in understanding the regions of functional importance on FKBP52, but we still do not know where the FKBP52 interaction site is on the receptors. The main hypothesis to be tested in this proposal is that FKBP52 potentiation of receptor function occurs through direct contacts between the FKBP52 FK1 domain and the receptor ligand binding domain leading to an enhancement of hormone binding. The dynamic nature by which the receptors achieve their hormone bound conformations to which FKBP52 associates makes it impractical to use a simple purified protein system. Thus, we must use more sophisticated approaches to analyze direct interactions between the steroid receptors and associated cochaperones. Each of the specific aims detailed below are independent from each other, but are all aimed at proving the proposed interaction between the FKBP52 FK1 domain and the receptor LBD and targeting that interaction with small molecule inhibitors. The long term goal of this project is the development of novel drugs for the treatment of hormone-dependent cancers. Towards this goal our immediate specific aims are: Aim #1: Use a previously validated androgen receptor-mediated reporter assay in yeast to identify random mutations within the receptor that alter the receptor's response to the FKBP proteins. Aim #2: Use directed photo affinity labeling of a cysteine-lacking FKBP52 mutant in combination with cross linking/mass spectrometry to identify and characterize the FKBP52-receptor interaction site. Aim #3: Screen compound libraries for selective FKBP52 inhibitors and characterize the specificity of inhibition and FKBP52 binding sites. We have identified two selective FKBP52 inhibitors to date.